Oxidative Stress Is Not a Major Contributor to Somatic Mitochondrial DNA Mutations

The accumulation of somatic mitochondrial DNA (mtDNA) mutations is implicated in aging and common diseases of the elderly, including cancer and neurodegenerative disease. However, the mechanisms that influence the frequency of somatic mtDNA mutations are poorly understood. To develop a simple invertebrate model system to address this matter, we used the Random Mutation Capture (RMC) assay to characterize the age-dependent frequency and distribution of mtDNA mutations in the fruit fly Drosophila melanogaster. Because oxidative stress is a major suspect in the age-dependent accumulation of somatic mtDNA mutations, we also used the RMC assay to explore the influence of oxidative stress on the somatic mtDNA mutation frequency. We found that many of the features associated with mtDNA mutations in vertebrates are conserved in Drosophila, including a comparable somatic mtDNA mutation frequency (similar to 10(-5)), an increased frequency of mtDNA mutations with age, and a prevalence of transition mutations. Only a small fraction of the mtDNA mutations detected in young or old animals were G : C to T : A transversions, a signature of oxidative damage, and loss-of-function mutations in the mitochondrial superoxide dismutase, Sod2, had no detectable influence on the somatic mtDNA mutation frequency. Moreover, a loss-of-function mutation in Ogg1, which encodes a DNA repair enzyme that removes oxidatively damaged deoxyguanosine residues (8-hydroxy-2 '-deoxyguanosine), did not significantly influence the somatic mtDNA mutation frequency of Sod2 mutants. Together, these findings indicate that oxidative stress is not a major cause of somatic mtDNA mutations. Our data instead suggests that somatic mtDNA mutations arise primarily from errors that occur during mtDNA replication. Further studies using Drosophila should aid in the identification of factors that influence the frequency of somatic mtDNA mutations. Author Summary Mitochondria are the evolutionary remnants of bacteria that were acquired by the cells of our ancestors more than a billion years ago and now produce virtually all of the cellular energy. Due to their bacterial ancestry, mitochondria have their own genomes, which encode some of the machinery responsible for producing energy. These genes occasionally acquire mutations-irreversible alterations that can adversely affect the energy production machinery. The accumulation of mitochondrial DNA (mtDNA) mutations is thought to cause aging and common age-related diseases, but we know little about the factors that influence the frequency of these mutations. Our study tested whether fruit flies would serve as a good animal model to study this problem. We found that flies accumulate mtDNA mutations in a pattern similar to that of humans. We then used flies to test the long-standing theory that toxic free radicals, chemical byproducts of energy production, cause mtDNA mutations to accumulate. Our data do not support this hypothesis, and instead suggest that rare errors associated with duplicating mitochondrial genomes are primarily responsible for mtDNA mutations. In sum we demonstrate that Drosophila serves as a tractable genetic model to investigate the mechanisms that influence the frequency of somatic mtDNA mutations.